Zootaxa 4306 (4): 580–592 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4306.4.8 http://zoobank.org/urn:lsid:zoobank.org:pub:3EC84E84-06D7-4B0C-8766-92842F768FED Descriptions of larvae of Vestalaria venusta (Hämäläinen, 2004) and Matrona basilaris Selys, 1853 (Odonata: Calopterygidae) RUNXI WANG1, XIN YU1, 2, JUNLI XUE1 & XIN NING1 1Insititute of Entomology, College of Life Sciences, Nankai University, Tianjin, 300071, China 2Corresponding author. E-mail: [email protected] Abstract Larva of Vestalaria venusta is identified using DNA barcoding match with the adult and described in the first time. Mor- phological characters are compared with those of Matrona basilaris and Vestalis amoena. The validity of genus the Ves- talaria is reconfirmed. The important role of DNA barcoding in odonate larva identification is emphasized. Key words: Zygoptera, Vestalaria, Matrona, Vestalis, larva, DNA barcode Introduction Vestalaria May, 1935, a small genus of Calopterygidae, is restricted to south China and Vietnam and includes five known species: V. m iao (Wilson & Reels, 2001), V. smaragdina (Selys, 1879), V. velata (Ris, 1912), V. venusta (Hämäläinen, 2004), V. vinnula Hämäläinen, 2006 (Hämäläinen 2016, Schorr & Paulson 2017). The genus Vestalaria was recently reinstated as separate from Vestalis Selys, 1853 by Hämäläinen (2004, 2006) according to careful morphological studies on adults and this received further support from a molecular study (Guan et al. 2012). No larva of Vestalaria has been described yet. DNA barcoding as a practical tool for associating larvae and adults of insects (Hebert et al. 2004), has been increasingly used in Odonata (Orr & Dow 2015, Steinhoff et al. 2016, Yu 2016). It has been confirmed that the combined nuclear gene of the ribosomal ITS1–5.8S–ITS2 region (ITS) is quite stable and reliable in molecular taxonomy at the genus and species level in Odonata, especially in Calopterygidae (Guan et al. 2012, Yu et al. 2015). In this study we use DNA barcoding, based on ITS sequence, to identify and describe a larva of genus Vestalaria. It is compared morphologically with Matrona and Vestalis larvae. Material and methods Samples. Calopterygidae adults and larvae were sampled from three localities (cf. Taxonomic account). Adults were caught with a standard insect net. Larvae were collected using a dip net and manual removal of stones. Attempts were made to rear the larvae in plastic containers but they died en route to the laboratory. Photographs of living specimens were taken in the field, before or just after collection, with a digital camera (Nikon D3200, Thailand). Photographs of detailed morphological features were taken in the laboratory using a Zeiss V20 microphotography system. Specimens were preserved in absolute ethanol and were examined and dissected under a Zeiss V8 stereomicroscope. Larvae from all localities were morphologically compared to each other and to description of the larva of Vestalis amoena Selys, 1853 given in Lieftinck (1965). As V. amoena larvae were not available for dissection and direct comparison to the sampled material in this study, this species was left out of the morphological characterization given below. Watson (1956) terminology of larva mandible description was used. At the time of collecting in 2011 (cf. Taxonomic account) the second author (XY) found a little stream where 580 Accepted by M. Marinov: 29 Jun. 2017; published: 18 Aug. 2017 the rare species Matrona oreades Hämäläinen, Yu & Zhang, 2011 was very abundant – both tenerals and sexually mature individuals. As no other calopterygid species was found around the stream (except a few of Archineura incarnata (Karsch, 1891) and one teneral male of V. venusta from more than 50 meters away) the larvae were provisionally identified as the dominant adult at the site M. oreades. However, according to subsequent molecular analysis published in Yu et al. (2015), the larvae were not Matrona but closely related to Vestalis, thus necessitating a special study of the larvae. In order to confirm the identification and classification of the larvae, ITS sequences from a total of 23 specimens were selected for the phylogenetic analysis (Table 1). Those sequences included material of the V. venusta and Matrona basilaris Selys, 1853 larvae described morphologically below in addition to already deposited nine sequences of Vestalis and Vestalaria species in NCBI (National Centre for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) and 11 sequences of Matrona species used in Yu et al. (2015). Note, that Yu et al. (2015) utilised the four M. basilaris used for the morphological description of larvae in this study. Voucher materials of all specimens except those from NCBI were deposited in the Institute of Entomology, College of Life Sciences, Nankai University, China. DNA extraction and sequencing. Total genomic DNA was isolated from hind leg muscle samples using UniversalGen DNA Kit (Beijing ComWin Biotech). Small doses were used as a template for polymerase chain reaction (PCR) amplification. PCR was carried out in 40 µL final reaction volumes containing 20µL of 2×Es Taq MasterMix (Beijing ComWin Biotech), 15.5µL ddH2O, 1.5µL of each primer and genomic DNA. For larvae samples, the DNA fragments of nuclear internal transcribed spacer (ITS) were amplified with the primers Vrain2F (5’-CTT TGT ACA CAC CGC CCG TCG CT-3’) and Vrain2R (5’-TTT CAC TCG CCG TTA CTA AGG GAA TC-3’) (Dumont et al. 2010). For the adult Vestalaria venusta, ITS 1 and ITS 2 were amplified with the following primers respectively: 5’-GGC CAA ACT TGA TCA TTT AG-3’ and 5’-GCC GGC CCT CAG CCA G-3’ for ITS1, 5’-CGG TGG ATC ACT CGG CTC GT-3’ and 5’-TTT CAC TCG CCG TTA CTA AGG GAA TC-3’ for ITS2 (Futahashi & Sasamoto 2012). The PCR cycling procedure was 2 min at 95°C followed by 35 cycles of denaturation at 95°C for 30s, annealing temperature at 56°C for 30s, and extension at 72°C for 1 min, with a final single extra extension step at 72°C for 8min (Larvae specimens). Annealing temperature 46.5°C for ITS1, 56.5°C for ITS2 for the adult specimen. All PCR products were visualized via 1% agarose gel electrophoresis and amplifications were purified using a gel extraction kit (Sangon Biotech, Shanghai), then sent to commercial companies (BGI, Beijing) for sequencing based on Sanger’s chain termination method. All fragments were sequenced in both directions. DNA analysis. All sequences were edited, assembled and aligned in BioEdit v7.2.0 (Hall 1999). Alignments of protein coding genes were translated to amino acids using MEGA v6.06 (Tamura et al. 2013) to detect frameshift mutations and premature stop codons, which may indicate the presence of pseudogenes. Sequences were aligned using the ClustalX version 2.1 program package (http://www.clustal.org/) with default settings, and subsequently corrected manually in terms of the sequence chromatogram to ensure each mutation loci was credible. Subsequent analyses were performed using optimality criteria including maximumlikelihood (ML) and the Bayesian inference algorithm (BI) to resolve the phylogenetic relationships. ML analyses were derived using MEGA v6.06 General Time Reversible model, predicted through ModelTest3.7 (Posada & Crandall 1998). BI analysis was performed using MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001) under GTR + I + G model implied by MrModeltest v2.3 (Nylander 2004). All the acquired trees were set to 1 million generations and for every 1000 generations the chain was sampled. Generations with standard deviation values higher than 0.01 were burned. Trees were displayed and rendered with FigTree v1.4.0 (Rambaut 2012). Results Based on the 23 sequences of ITS, all the phylogenetic trees (ML, BI) produced the same topological results, which strongly supported (BPP=0.84, MLB=95) the larvae and the adult V. venusta to form a monophylum (Fig. 1). Vestalaria was also recovered as a monophylum (BPP=1.00, MLB=100) with Vestalis as sister group with very strong support (BPP=1.00, MLB=100). In the Matrona clade, M. basilaris together with M. mazu Yu, Xue & Hämäläinen, 2015 formed a monophylum (BPP=1.00, MLB=95) with M. oreades as sister (BPP=1.00, MLB=100). This result indicated that the larvae involved here are the same species with the adult, viz. V. venusta. The validity of genus level of Vestalaria was confirmed again by molecular phylogeny. LARVA OF VESTALARIA VENUSTA Zootaxa 4306 (4) © 2017 Magnolia Press · 581 582 · Zootaxa 4306 (4) ©2017 (4) 4306 Vestalis beryllae ! "# $ %& ' Vestalis gracilis ()*+*, "# -$ . /& 0 Vestalis atropha 2 $ " $ 0&& 3 & 4 Vestalis amaryllis 2 $ " $ 0&& 3 & 4 Magnolia Press Vestalis anne ()+ , %5$ . /& 0 Vestalis amoena ()+ ) "#$ " 6 $ . /& 0 Vestalis lugens ()+ * $ 7 8 -&$ /& 0 Vestalis smaragdina ()+ ! 5 /$ $ . /& 0 Vestalaria velata 2 9 5 5$ 0&& Vestalaria venusta :(* :$ 6-$ ! , ; : Vestalaria venusta -< :() :$ 6-$ ! ) ; : Vestalaria venusta -< :(, :$ 6-$ ! ) ; : Matrona basilaris -< : et al. = )> (0() $ ( 6-$ ) 05? .5 Matrona basilaris -< : et al. = )> (0(+ $ ( 6-$ ) ; : Matrona basilaris -< : et al. = )> (0( $ ( 6-$ ) ; : Matrona basilaris -< : et al. = )> (0( $ ( 6-$ ) 05? .5 Matrona mazu : et al. = )> 07@, 7 A $ 0 6-$ !)! ; : Matrona mazu : et al. = )> 07@! 7 A $ 0 6-$ !)! ; : Matrona oreades B : et al. = )> 97; 7C$ 9 6-$ )! ; : Matrona oreades : et al. = )> 97; 7C$ 9 6-$ )! ; : Matrona oreades : et al. = )> 97; 7C$ 9 6-$ )! ; : Matrona oreades : et al. = )> :( :$ 6-$ ! , ; : Matrona oreades : et al. = )> :( :$ 6-$ ! , ; : WANG WANG ET AL. FIGURE 1. Phylogenetic reconstruction based on ITS. Values of Bayesian posterior probabilities and Maximum likelihood bootstrap are indicated at nodes respectively, the adult and larvae of Vestalaria venusta are in red colour. Taxonomic account Vestalaria venusta Hämäläinen, 2004 Material studied. Adult 1 ♂ (SCYA08) and larvae 1 ♂ (SCYA05), 1 ♀ (SCYA06): China, Sichuan, Yaan, Bifengxia Town, Houyan Village (103.0472° E, 30.1019° N), 25-vii-2011, Xin Yu leg. Description of larva.